The aim of this proposal is to establish a modern pulsed Electron-Nuclear Double Resonance (ENDOR) capability at Cornell University to be located in the National Biomedical Center for Advanced ESR Technology (ACERT). It will be used by research groups at Cornell, University of Rochester and SUNY Buffalo. ACERT possesses all the necessary expertise and technical capability to maintain the facility. The NIH-supported research projects of the major users include: (1) ENDOR study of novel chemistry of a [4Fe-4S]-cluster- containing radical enzyme in diphthamide biosynthesis; (2) ENDOR and Electron-Spin Echo Envelope Modulation (ESEEM) to probe the role of hydration in membrane fusion; (3) ENDOR spectroscopy studies of proteins that mediate signal transduction through redox and photochemistry; (4) mechanism of inhibition of ribonuclease reductase by anti-cancer drugs; and (5) structure and dynamics of porphysomes - new photo- sensitive agents in cancer therapy and drug delivery. Additional projects from minor users include: (6) structure and function of artificial hydrogenases; (7) nitrification of enzymes and electron transfer proteins; and (8) hydration at lipid surfaces. ACERT, since its establishment in 2001 provides EPR expertise and service to many research groups nationally which represent many biomedical fields. While the EPR currently performed in ACERT has been crucial in obtaining structural and functional information on paramagnetic cofactors in biological systems, such as iron- sulfur clusters, copper active sites etc., additional studies using ENDOR were often necessary but not available at Cornell for these users. Having an ENDOR shared capability at Cornell to be housed in ACERT will dramatically improve the quality of research for the groups participating in this proposal, as well as others who interact with ACERT. They will have control of their ENDOR measurements, which would become part of their daily research. Also, importantly, ENDOR and EPR studies will be carried out at the same time on the same samples and under the same conditions. As described in projects of major/minor users, ENDOR and EPR (e.g. ESEEM) are complimentary to each other in terms of revealing different aspects of interactions between the unpaired electron and nuclear spins present in the biological system studied.